The antigenic structure of dengue envelope (E) glycoprotein was investigated with the goal of increasing its immunogenicity. Recombinant vaccinia viruses were constructed that contained dengue cDNA coding for C-terminally truncated E that ranged in length from 9% to 99% of the N-terminal sequence. Overall antigenicity of the E products was analyzed by radio-immunoprecipitation using dengue hyperimmune mouse ascitic fluid HMAF) or an E peptide antiserum. With the exception of 59%E, only E constructs greater than 79% were able to bind HMAF efficiently. Arg at position 392 in the dengue type 4 E sequence immediately following the 79%E C-terminus appeared to be critical for proper conformation and antigenic structure required for efficient binding by HMAF. Full-length E was retained intracellularly, and only truncated E ranging from 59% to 81% in length was secreted extracellularly. Only 79%E-RKG (that possessed the 3 additional amino acids immediately downstream of 79%E) was expressed in high concentration on the surface of recombinant virus-infected cells presumably being inserted into the plasma membrane by a C-terminal membrane anchor that included a 20 amino acid hydrophobic transmembrane domain and a short charged amino acid cytoplasmic domain. Studies in mice indicated that only truncated E's that were recognized efficiently by HMAF induced a high level of resistance to dengue virus encephalitis. Among the truncated E's that were able to bind HMAF efficiently only 79%E-RKG was expressed in high concentration on the cell surface. Significantly, only 79%E-RKG induced an appreciable E antibody response suggesting that cell surface expression was responsible for its enhanced immunogenicity. Finally, passive immunization studies in mice involving serum transfer indicated that serum antibodies to E played a major role in the complete or nearly complete resistance to dengue virus challenge induced by certain vaccinia virus-truncated E recombinants.